Objective:To investigate the change of the expression of Cyclin D1 and Cyclin E in astrocytes of rats after focal cerebral ischemia.Methods:Ischemia was induced by temporary middle cerebral artery occlusion(MCAO).The rats suffered from MCAO were sacrificed on days 1,3,7 or 15 after reperfusion and rats in control group were sacrificed on day 1 after surgical operation.The brain was taken and the expressions of Cyclin D1 and Cyclin E in astrocytes were detected by flow cytometry.Results:The expressions of Cyclin D1 and Cyclin E were detected in astrocytes of the controls and the expression of Cyclin E was higher than that of Cyclin D1(P0.05).After focal cerebral ischemia,the expression of Cyclin D1 in astrocytes was significantly increased on the edge of ischemic core at 7 and 15 days after reperfusion(P0.05),while the expression of Cyclin E had no significant variation.Conclusion:Cyclin D1 may take part in the progression of the cell cycle in G1/S transition in proliferating astrocytes.

Objective:To investigate the effect of X-ray on proliferation of cultured astrocytes(AS) and the action mechanism.Methods: The 80% confluent cultured AS were irradiated by different doses of X-ray,and effects of irradiation on cell cycle and apoptosis were analyzed by flow cytometry.The 80% confluent cultured AS were synchronized by serum deprivation for 48 h,then the cultured AS were randomly divided into 2 groups: control group(10% FBS) and irradiation group(10% FBS+irradiation).Western bolt was used to detect the expression of PCNA,p53 and cyclin E.Results:X-ray irradiation could reduce the percentage of cells in S phase in a dose-dependent manner,but not increase the apoptosis rate of AS.After the culture was serum-starved for 24 h,almost confluent AS remained at G0 phase.As a response to FBS,the percent of S phase cells and the expression levels of PCNA and cyclin E were increased in control group;In irradiation group,the percent of S phase cells was decreased dose-dependently.The expression of PCNA and cyclin E was also inhibited significantly,and the expression of p53 increased significantly in irradiation group as compared with control group.Conclusion: X-ray can effectively inhibit proliferation of AS by regulating cell cycle of AS.

Objective To study the change in the expressions of astrocytes and neurons of rats after focal cerebral ischemia and the difference in the expression of cyclin D1,respectively.Methods Ischemia was induced by temporary middle cerebral artery occlusion(MCAO).The experimental rats suffering from MCAO were sacrificed on days 1,3,7 and 14 after reperfusion.The brain was taken and the expressions of astrocytes and neurons,and cyclin D1 expression in astrocytes and neurons were respectively detected by flow cytometry.Results Compared with those of the controls the expression of astrocytes was remarkably up-regulated on the side of ischemia(P<0.05),while the expression of neurons was decreased(P<0.05);and the expression of cyclin D1 in astrocytes and neurons on the side of ischemia was up-regulated,expecially in astrocytes after 7 and 14 days of cerebral ischemia/reperfusion(P<0.05).Conclusion After focal cerebral ischemia,astrocytes and neurons have an up-regulated of cyclin D1 expression.

Objective To determine the effect of olomoucine inhibitor of cyclin-dependent protein kinases on the activation and proliferation of cultured astrocyte after mechanical injury.Methods The model of mechanical injury on cultured purified astrocytes was established.The cultured cells were randomly divided into 3 groups:control group,scratch group(scratch + vehicle),and olomoucine group(scratch + olomoucine).Immunofluorescence(IF)staining was used to detect the expression of glial fibrillary acidic protein(GFAP).RT-PCR was used to detect the mRNA level of IL-6 and TNF-α.Results The "scratch-wound injury" induced AS proliferation and activation.In the early phase of scratch injury,the mRNA level of IL-6 and TNF-α increased.From 12 h after injury,AS around the injury area showed hypertrophy and proliferation.In the olomoucine group,the number of GFAP positive cells decreased significantly.Moreover,olomoucine inhibited the expression of IL-6 and TNF-α.Conclusion As a response to injury,AS enter the cell cycle and showed reactive gliosis.The cell cycle inhibitor olomoucine can inhibit injury-induced reactive gliosis and astrocyte activation by regulating the cell cycle.

Objective: To construct Flag-p27 and EGFP co-expressed pNSE-IRES2-EGFP-p27 plasmid,and to detect its expression in cultivated neurons.Methods: By way of genetic engineering techniques as plasmid extraction,agarose gel electrophoresis,restriction enzymolysis,connection,transformation of competent cells,the recombinant plasmid of pNSE-IRES2-EGFP-p27 was obtained.The constructed plasmids were transfected to cultivated neurons with liposome and the expression of EGFP and p27 was detected by immunocytochemistry.Results:pNSE-IRES2-EGFP-p27 was successfully constructed and verified by enzymolysis and sequencing.Confocal microscopy revealed that after the plasmids were transfected to neurons,the expression of EGFP could be detected and the expression of p27 was increased significantly in EGFP positive cells.Conclusion:The vectors containing the NSE promoter resulted in the EGFP and p27 expression in neurons.EGFP can be as a report gene to indicate the expression of p27.The construction of pNSE-IRES2-EGFP-p27 lies a foundation for future research on the relationship between cell cycle of neurons and nervous system disease.

OBJECTIVE:To investigate the effects of olomoucine, a cyclin dependent protein kinase (CDK) inhibitor, on the neuronal apoptosis after status epilepticus (SE). METHODS:Lithium chloride was injected intraperitoneally, and pilocarpine was injected intraperitoneally after 18 h to 24 SD rats so as to cause SE. Twenty-two of the 24 rats developed SE and 2 of them died. The surviving 20 rats were then randomly divided into 2 equal groups: olomoucine group, injected intracerebroventricularly after the SE was terminated by diazepam and chloral hydrate once a day for 3 days, and SE group, infused intracerebroventricularly with DMSO solution Another 10 rats were injected intraperitoneally with normal saline and then infused intracerebroventricularly with DMSO solution to be used as control group. Six hours after SE attack 5 rats from each group were killed respectively with their brains taken out. Semiquantitative RT-PCR was used to detect the mRNA expression of anti-inflammatory cytokines, such as interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. Three days later the other 5 rats in each group were killed with their entorhinal cortex and hippocampus taken out. TUNEL was used to observe the apoptosis. Immunofluorescence (IF) staining was used to detect the expression of neuronal nuclear nucleoprotein (NeuN) and cyclin B1. RESULTS:TUNEL showed that apoptotic neurons were rare in the control group and were numerous in the SE group, especially in the entorhinal cortex and the hylus of dentate gyrus, and the number of apoptotic neurons in the hylus of dentate gyrus of the olomoucine group was not significantly different from that of the control group (P < 0.05), however, the number of apoptotic cells in the entorhinal cortex of the olomoucine group was still significantly higher than that of the control group (P < 0.05). IF staining demonstrated that in the control group the co-expression of NeuN and TUNEL-labeled cells was weak; and in the SE group the co-expression of NeuN and TUNEL was significantly increased compared with that in the control group (P < 0. 05). The number of cyclin B1 positive cells in the olomoucine group was 18.22 +/- 3.99, significantly lower than that of the SE group (24.57 +/- 6.78, P < 0.05). Semiquantitative RT-PCR showed that the IL-1beta and TNF-alpha mRNA expression levels of the SE group were both significantly higher than those of the control group (both P < 0.05), and the IL-1beta and TNF-alpha mRNA expression levels of the olomoucine group, except the TNF-alpha mRNA expression in the cortex, were all significantly lower than those of the SE group (all P < 0.05), and not significantly different from those of the control group (all P > 0.05). CONCLUSION:Olomoucine treatment can inhibits cell cycle protein B1 expression, anti-inflammatory cytokines such as IL-1beta and TNF-alpha secretion, thus decreasing neuronal death and providing neuroprotection after SE, which suggests a potential promising therapeutic way for epilepsy treatment.

Objective To study the change of expression of P21cip1 in astrocytes and neurons of rats after focal cerebral ischemia.Methods Ischemia was induced by temporary middle cerebral artery occlusion(MCAo).The experimental rats suffered from MCAo were sacrificed on days 1,3,7 and 14 after reperfusion.The brain was taken and the P21cip1of astrocytes and neurons was detected by flow cytometry.Results Compared with that of the controls,the expression of P21cip1 in astrocytes and neurons was remarkably down-regulated on tie ischemic side of cerebral cortex after 3,7 and 14 days of cerebral ischemia/reperfusion(P< 0.05).The P21cip1 expression showed no marked difference between astrocytes and neurons(P> 0.05).Conclusion Astrocytes and neurons have a downregulation of expression of P21cip1after focal cerebral ischemia.

Objective:The change of expression of p15~(ink4b) on astrocytes and neurons of rats after focal cerebral ischemia has been stuided.Methods: Ischemia was induced by temporary middle cerebral artery occlusion(MCAO).The experimental rats induced by MCAO were sacrificed on days 1,3,7 and 14 after reperfusion.The brain was taken at days 1,3,7,14 after injury,then the p15~(ink4b) of astrocytes and neurons was detected by flow cytometry.Results: Compared with that of the controls,the expression of p15~(ink4b) on astrocytes was remarkably down-regulated on the ischemic side of cerebral cortex 3,7 and 14 days after reperfusion(P 0.05).Similarly,p15~(ink4b) on neurons was significantly down-regulated on the ischemic region 14 days after cerebral ischemia(P 0.05),but lesser than that on the astrocytes(P 0.05).Conclusion: Astrocytes and neurons show a different down-regulated expression of p15~(ink4b) after MCAO in rats.The p15~(ink4b) expression on astrocytes was more remarkably down-regulated than that on neurons.